Phosphinic pseudo-peptides that may be used as matrix zinc metalloprotease inhibitors

ABSTRACT

The invention relates to pseudo-peptides according to the formula:                    
     wherein 
     R 1  is a group inhibiting an amine function, or an amino acid residue or peptide with an inhibited terminal amino function, 
     R 2  represents the lateral chain of a natural or non-natural amino acid, 
     R 3  represents: 
     1) the lateral chain of a natural amino acid except for Gly and Ala, not substituted or substituted by an aryl group, 
     2) an aralkyl group, or 
     3) an alkyl group comprising at least 3 carbon atoms, and 
     R 4  represents a lateral chain of natural or non-natural amino acid. 
     They are useful as matrix zinc metalloprotease inhibitors, particularly in the treatment of cancer.

This application is a 371 of PCT/FR00/00093 Jan. 18, 2000.

FIELD OF THE INVENTION

The present invention relates to phosphinic pseudo-peptides that mayparticularly be used as very powerful and specific inhibitors for matrixzinc metalloproteases MMP, particularly MMP-11, MMP-2, MMP-9 and MMP-8.However, these pseudo-peptides appear to show a low activity withrespect to MMP-1 and MMP-7. In this way, these inhibitors offer thepossibility to only inhibit one MMP sub-family in vivo, and couldtherefore prove to be less toxic than MMP inhibitors with a very broadspectrum of activity.

Said pseudo-peptides may have applications in the treatment of diseasescharacterised by the over-expression of matrix proteases, such asconnective and joint tissue degeneration, rheumatoid arthritis,osteoarthritis, aortic aneurysm and cancer.

On the basis of the studies conducted in vivo on animals, thesephosphinic inhibitors appear to allow use in pharmaceutical formulationsto inhibit primary or secondary tumour growth.

STATE OF THE RELATED ART

Matrix zinc metalloproteases MMP represent a family of enzymescollectively capable of splitting all the proteins of the extracellularmatrix. Due to their role on extracellular matrix proteins, theseenzymes play a very important role during the development and the courseof various tissue remodelling processes, such as involution of themammary gland, cicatrisation and extravasion of specialised immuneresponse cells.

Around fifteen enzymes in humans belonging to the MMP family are knownto date:

Matrixins MMP-1 Interstitial collagenase MMP-8 Neutrophil collagenaseMMP-13 Collagenase 3 MMP-18 Collagenase 4 MMP-3 Stromelysin 1 MMP-10Stromelysin-2 MMP-11 Stromelysin-3 MMP-2 Gelatinase-A MMP-9 Gelatinase-BMMP-12 Metalloelastase MMP-14 MT-1 MMP MMP-15 MT-2 MMP MMP-16 MT-3 MMPMMP-17 MT-4 MMP MMP-7 Matrilysin

Collagenases appear to be the only MMPs to be capable of splittingcollagen in fibrillary form. Gelatinases A and B are characterised bytheir ability to split type IV collagen, which is very abundant in thebasal membranes and collagen in denatured form. Stromelysins 1 and 2appear to be responsible for the breakdown of other proteins of theextracellular matrix, such as fibronectin and various proteoglycans.MT-MMPs appear to be above all involved in the activation of gelatinaseA, and due to their membrane location, these matrixins appear to play arole as a membrane receptor of gelatinase A. It is important to notethat the physiological substrate of stromelysin-3 is not known to date.However, several studies on this protease, which was initiallycharacterised in breast tumours, suggest that stromelysin-3 is animportant factor in the development and survival of tumours.

MMPs appear to be over-expressed in various human diseases, particularlycancer. In this disease, for a long time, the role of MMPs wasassociated with the invasion of tumour cells and their ability to passthrough various barriers to form secondary tumours. More recently,various studies have established that said proteases certainly play amore fundamental role in carcinogenesis, particularly by taking part inprimary tumour growth. Of different theories to explain this function ofMMPs, the most studied relate to their role in:

their ability, by means of extracellular matrix protein proteolysis, torelease from said matrix the growth factors essential for thedevelopment and survival of tumours, and

angiogenesis, required for tumour growth.

The apparent involvement of MMPs in tumour growth has led numerous teamsworld-wide to focus on the role of compounds capable of inhibiting theseenzymes.

Synthesis programmes on MMP inhibitors were initiated a number of yearsago. At this time, applications of MMP inhibitors particularly relatedto inflammatory diseases of the connective tissue. It was only morerecently that multiple programmes on the application of MMP inhibitorsin cancerology have developed, as described by Brown, Medical Oncology,1997, 14, 1-10, [1]. Indeed, as mentioned above, the studiesdemonstrating that MMPs must be considered as priority targets in thedevelopment of new anticancer agents are relatively recent. As such, itmay be noted that, in 1997, 67 patents relating to MMP inhibitors wereregistered world-wide, the majority of which relate to cancerologyapplications, as described by Beckett et al, 1998, Exp. Opin. Ther.Patents, 8, p.259-289 [2]. In most of these patents, the synthesisedcompounds belong to the family of pseudo-peptide derivatives comprisinga hydroxamate function. Some patents relate to pseudo-peptide compoundscomprising carboxyl-alkyl or thiol functions. In said compounds, thehydroxamate, thiol or carboxyl-alkyl functions interact with the zincatom present in the active MMP site. The most advanced compounds interms of anticancer activity have been developed by the firm BritishBiotechnology. Two compounds, Batimastat BB94 and Marimastat have beenthe subject of phase II and III clinical studies in humans. Since then,other firms (Roche, Bayer, Agouron, Novartis) appear to have conductedphase I and II clinical studies on MMP inhibitors in cancerology.

In this way, none of the compounds liable to inhibit MMPs known by meansof references [1] and [2], are composed of a phosphinic pseudo-peptide.

However, the document: Goulet et al, Bioorg. Med. Chem. Lett. 4, 1994,p. 1221-1224 [3], describes phosphinic pseudo-peptides that can be usedas a selective inhibitor of stromelysin-1 (MMP-3), which comprise thefinal sequence:

Caldwell et al, Bioorg. Med. Chem. Letter 6, 1996, p.323-328 [4], alsodescribe a phosphinic pseudo-peptide which is a selective inhibitor ofstromelysin-1 (MMP-3), which comprises the same terminal sequence as thepseudo-peptide of reference [3].

In these references [3] and [4], it is attempted to detect activity withrespect to MMP-3, while in the invention, it is attempted to detect aselective activity with respect to MMP-11, MMP-2, MMP-9 and MMP-8. It isalso important to note that, in the present invention, R³ is not only aphenylethyl residue. In addition, the invention demonstrates that othersubstituents in this position give compounds with increased inhibitorypower.

The documents FR-A-2 676 059 [5], FR-A-2 689 764 [6] and EP-A-0 725075(7] illustrate phosphinic pseudo-peptides showing an inhibitoryactivity with respect to bacterial collagenases and zinc endopeptidases24.15 and 24.16.

The present invention specifically relates to new phosphinicpseudo-peptides showing a powerful and specific inhibitory activity withrespect to the matrix zinc metalloproteases MMP-11, MMP-2, MMP-9 andMMP-8.

DESCRIPTION OF THE INVENTION

According to the invention, the phosphinic pseudo-peptide complies withthe formula:

wherein

R¹ is a group inhibiting an amine function, or an amino acid residue orpeptide with an amino-terminal group protecting function,

R² represents the lateral chain of a natural or non-natural amino acid,

R³ represents:

1) the lateral chain of a natural amino acid except for Gly and Ala, notsubstituted or substituted by an aryl group,

2) an aralkyl group, or

3) an alkyl group comprising at least 3 carbon atoms, and

R⁴ represents a lateral chain of natural or non-natural amino acid, or adinitrobenzyl group.

Said pseudo-peptide according to formula I is a pseudo-tripeptidecomprising a phosphinic type chemical group, the function of which is tochelate the zinc atom in MMPs. In said pseudo-tripeptide, the choice ofthe R², R³ and R⁴ groups makes it possible to ensure the interaction ofthe tripeptide with the MMP sub-sites S1, S1′ and S2′, respectively. TheR¹ group is assumed to interact at the junction of the sub-sites S3/S2.

In the above definition of the pseudo-peptides according to theinvention, the terms “amino acid” used for R¹, R², R³ and R⁴ in [5]refer to the twenty α-amino acids commonly found in proteins which arealso known as standard amino acids and their analogues. The lateralchains of said amino acids comprise linear and ramified alkyl,hydroxyalkyl, carboxyalkyl, aralkyl, aminoalkyl, carboxamide alkyl,mercapto alkyl, phenylalkyl, hydroxyphenylalkyl, guanidinoalkyl,imidazoylalkyl, indolylalkyl, and pyrrolidinyl groups.

Examples of amino acid that may be used include alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, iso-leucine, leucine, norleucine, lysin, methionine,phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan,tyrosine, valine, nitrophenylalanine, homo-arginine, thiazolidine anddehydroproline.

However, in the case of R³, the amino acid cannot be Gly or Ala sincethey do not show sufficient interaction with the MMP sub-site S1′.

Preferentially, the amino acid used for R³ is chosen from Phe, Leu orSer, Cys residues wherein the lateral chain is substituted by anarylalkyl group.

The aryl groups liable to be used are those derived from a monocyclic orpolycyclic aromatic core, possibly substituted by alkyl or alcoxygroups. Examples of aryl groups that may be used include the phenyl,naphthyl, benzyl and alcoxybenzyl groups, such as p-methoxybenzyl.

R³ may also represent an aralkyl group. In said aralkyl group, the arylgroup may be any of the other mentioned above. The alkyl part of thearalkyl group may be a linear or ramified chain of 1 to 6 carbon atoms.

Examples of the aralkyl groups that may be used include groups accordingto the formulas:

where n is an integer from 1 to 4, and

where p is equal to 1 or 2.

The alkyl groups that may be used for R³ have at least 3 carbon atoms.They may be linear or ramified and preferentially have at most 7 carbonatoms. Examples of alkyl groups that may be used include the groupsCH₃—(CH₂)₆— and (CH₃)₂—CH—CH₂—.

Preferentially, R³ represents a group complying of any of the followingformulas:

According to the invention, R² is chosen so as to interact with the MMPsub-site S1. Good results are obtained when R² represents the methyl orbenzyl group; preferentially, R² is the benzyl group, which correspondsto the lateral chain of Phe.

According to the invention, R⁴ is chosen so as to interact with the MMPsub-site S2′. Good results are obtained when R⁴ represents the lateralchain of Trp or a dinitrobenzyl group Dpa.

In the pseudo-peptide according to the invention, the lateral chains R²,R³ and R⁴ of the amino acids may be in L or D form. In addition, thepseudo-peptide may be composed of a single isomer or by a mixture of4-diasteroisomers due to the presence of two asymmetrical centres on theα carbon comprising the R² and R³ residues. Although any amino acidconfiguration may be suitable, it is preferable that the unit:

has an L configuration.

However, three out of four diastereoisomers corresponding to differentR² and R³ configurations have practically equivalent activities as MMPinhibitors.

In the pseudo-peptides according to the invention, R¹ may representvarious groups, the nature of which influences the affinity of thepseudo-peptide with respect to the different MMPs.

R¹ may represent a “group inhibiting an amine function”. These termsinclude all the inhibiting groups that may be used to inhibit the aminefunctions of amino acids and peptides, for example t-butoxy-carbonyl,benzyloxycarbonyl, cinnamoyl, pivalolyl andN-(I-fluorenyl-methoxycarbonyl) Fmoc groups.

R¹ may also represent inhibiting groups chosen from the acetyl,benzyloxyacetyl, phenylaminoacetyl, (m-chlorophenyl)aminoacetyl,(2-hydroxy-5-chloro-phenyl) amino acetyl, indolyl-2-carbonyl,4,6-dichloro-indolyl-2-carbonyl, quinolyl-2-carbonyl and1-oxa-2,4-dichloro-7-naphthalene carbonyl groups, or an amino acid orpeptide residue wherein the terminal amine function is inhibited by asuitable group. Examples of such residues include the Z-Ala and Z-Leugroups wherein Z represents the benzyloxycarbonyl group.

The pseudo-peptides according to the invention may be prepared usingconventional methods from phosphinic blocks according to the formula:

wherein Z represents the benzyloxycarbonyl group and Ad the adamantylgroup, and the amino acid corresponding to R⁴ by solid phase chemicalsynthesis according to the methods described by Yotakis et al, J. Org.Chem., 1996, 61, page 6601-6605 [8] and Jiracek et al, J. Biol. Chem.,1995, 270, p. 21701-21706 [9] and J. Biol. Chem., 1996, 271, p.19606-19611 [10].

The initial phosphinic blocks may be obtained by Michael addition of aphosphinic acid comprising the R² group

on an acrylate supplying the R³ group

where Et represents the ethyl group.

The acrylates supplying the R³ group may be synthesised using differentprocesses, as seen below.

The pseudo-peptides according to the invention may be used in thetreatment of diseases involving an over-expression of matrix zincmetalloproteases.

The invention also relates to a pharmaceutical formulation inhibiting atleast one matrix zinc metalloprotease, comprising at least onepseudo-peptide according to formula I as defined above.

Preferentially, said formulation inhibits a matrix zinc metalloproteasechosen from MMP-2, MMP-8, MMP-9 and MMP-11.

Said formulation is intended to treat a disease characterised by theover-expression of matrix proteases, such as cancer.

The invention also relates to the use of a phosphinic pseudo-peptideaccording to formula I as defined above, to manufacture a medicinalproduct inhibiting at least one matrix zinc metalloprotease,particularly MMP-2, MMP-8, MMP-9 and MMP-11.

The invention's other characteristics and benefits will be seen moreclearly upon reading the following description, naturally given as anon-restrictive illustration, in relation to the appended figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a synthesis diagram of pseudo-peptides according to theinvention.

FIG. 2 is a diagram illustrating the anti-tumoral efficacy of a compoundaccording to the invention, which gives the mean volume of the tumour(in mm³) as a function of time (in days) after injecting cancerous cellsin control mice and mice treated with the pseudo-peptide according tothe invention.

DETAILED DESCRIPTION OF EMBODIMENTS

Examples 1 to 27 below illustrate the production of pseudo-peptidesaccording to the invention.

FIG. 1 represents the synthesis diagram of the pseudo-peptides accordingto the invention, which involves a first reaction to produce phosphinicblocks 3 from a phosphinic acid 1 comprising the R² group and acrylate 2supplying the R³ group, by means of Michael addition.

After the formation of the phosphinic block 3, an adamantyl group isintroduced on the hydroxyphosphinyl function of the block 4, and theC-terminal ester function 5 is then eliminated, and solid phasesynthesis of the pseudo-peptide is performed by adding the requiredamino acid 6 on the C-terminal acid function.

In this figure, Z represents the benzyloxycarbonyl group.

EXAMPLES 1 to 5

These examples illustrate the preparation of acrylates 2 comprising oneR³ group with terminal CH₂ using the following method:

This synthesis corresponds to the method described by Eistetter et al inJ. Med. Chem., 25, p109-113, 1982 [11].

This synthesis is described below in the case where R³ represents thegroups illustrated in table 1.

To a solution of sodium ethoxide (10 mmol of sodium in 11 ml of pureethanol), over a period of 10 minutes, 10 mmol of diethyl malonate isadded. The solution is placed under stirring at 50° C., for 1 hour, and10 mmol of the required bromide is then added drop by drop. The mixtureis placed under stirring under stirring for 6 hours at 50° C., and theethanol is eliminated in a vacuum and diethyl ether is added. Thissolution is washed with water, brine and dried on Na₂SO₄, and thenconcentrated to obtain an oily residue, which after distillation givesthe diester according to the formula:

with 40 to 65% yields.

To a 10 mmol diester solution in 8 ml of ethanol, a KOH solution (10mmol) in 8 ml of ethanol is added, and the mixture is stirred for 16hours. After the organic solvent has evaporated, the residue is treatedwith water and extracted with diethyl ether.

The aqueous phase is acidified with 6N HCl and extracted twice withdiethyl ether. The organic phases are dried on Na₂SO₄ and evaporated toobtain monoesters according to the formula:

with 65 to 90% yields.

To a monoester solution in 0.8 ml of pyridine and 0.05 ml of piperidine,6 mmol of paraformaldehyde is added, and the mixture is then placedunder stirring and heating at 50-55° C. for 3 hours. After addingdiethyl ether, the organic phase is washed with water and 3N HCl, andthen dried on Na₂SO₄, and concentrated to obtain compounds 2a to 2c inthe table with the yields indicated in said table.

The melting points of the acrylates 2a to 2e obtained are also given intable 1.

EXAMPLE 6

In this example where R³ represents CH₂R′³, the acrylate is synthesisedaccording to the following synthesis process:

In this example, this synthesis process is used to prepare the acrylate2 wherein R³ represents the CH₃(CH₂)₆ group, i.e. R′³═CH₃—(CH₂)₅. In anargon atmosphere, a hexyl bromide solution CH₃(CH₂)₅ Br(1.65 g, 10 mmol)in 15 ml of pure diethyl ether is added drop by drop to a flaskcontaining 0.22 g (11 mmol) of magnesium and iodine I₂ (catalytic) overa 90 minute period.

The reaction mixture is reflux boiled for 1 hour. After adding 0.18 mmolof CuI, the temperature of the mixture is decreased to −78° C.

A solution of 1.15 g (6.7 mmol) of the compound is then added slowly:

in 10 ml of Et₂O: THF. The mixture is stirred for 30 minutes at ambienttemperature. After treating the mixture with 0.5N HCl, 5% NaHCO₃ andwater, the organic layer is dried on Na₂SO₄. The solvent is eliminatedin a vacuum and the residue obtained is purified by columnchromatography using a 40 to 60° C. petroleum ether/ether mixture (13:1)as the eluent. The compound 2f is obtained in this way with a 40% yield.The characteristics of compound 2f are given in table 1.

EXAMPLE 7

In this example, an acrylate 2 is prepared with R₃═R′³—CH₂— using themethod described by Baldwin et al J. Chem. Soc. Chem. Comm. 1986, p1339-1340, [12].

This corresponds to the following reaction diagram:

This method is used to prepare the acrylate 2 g wherein R³ representsthe group according to the formula:

A mixture of 1.9 g (10 mmol) of ethyl bromomethyl acrylate and 3.3 g (20mmol) of sulphinic benzene acid sodium salt in 40 ml of methanol isreflux boiled for 12 hours. The solvent is eliminated and diethyl etheris added. The solution is washed with water, brine, dried on Na₂SO₄ andconcentrated to obtain 2 grams of sulphinic acrylate

in oil form, with an 80% yield.

To a mixture of 2 g (8 mmol) of sulphinic acrylate in 40 ml of drybenzene, 4.7 g (16.4 mmol) of stannic tri-n-butyl hydride and 0.15 g(0.96 mmol) of 2,2′-azobisisobutyronitrile (AIBN) are added. The mixtureis reflux boiled for 1.5 hours, and water is then added and the organiclayer extracted is dried on Na₂SO₄ and concentrated. The unprocessedproduct is purified by column chromatography, using petroleum ether (40to 60° C.)/ether (9:1) as an eluent. 2.9 g of alkylstannane is obtained

with a 90% yield.

1.47 g (3.6 mmol) of alkylstannane and 0.39 g (1.8 mmol) of 2-bromoethylnaphthalene are dissolved in 10 ml of dry benzene. After adding 0.064 g(0.39 mmol) of AIBN, the reaction mixture is reflux boiled for 2 hours.After adding water, the organic phase is separated, washed with waterdried on Na₂SO₄ and concentrated to dryness. In this way, 0.06 g of theacrylate 2 g given in table 1 is obtained, with a 13% yield, Afterpurifying the residue on a chromatography column using petroleum ether(40 to 60° C.)/ether (9:1) as an eluent.

EXAMPLES 8 AND 9

In these examples, an acrylate 2 is prepared, wherein R³ comprises asulphur atom or an oxygen atom, according to the following synthesisdiagram:

In this diagram, R³ corresponds to RX—CH₂ where X═S or O.

The method is used to prepare the acrylates 2h and 2i wherein R³represents the paramethoxybenzyl-thiomethyl group and thebenzylthiomethyl group, respectively.

A 10 mmol solution of suitable thiol R—SH in 15 ml of methanol is addeddrop by drop to a stirred solution cooled in an ice-bath of 9 mmol ofsodium in 20 ml of methanol over a 30 minute period. After adding thiol,the mixture is concentrated to dryness and diethyl ether is added. Thesalt which precipitates is cooled in an ice-bath. The product isfiltered, washed with cold Et₂O and dried on P₂O₅ to obtain the sodiumsalt with a 85 to 95% yield. To a mixture of 10 mmol of sodium salt insuspension in 40 ml of dry Et₂O and cooled in an ice-bath, over a 45minute period, a solution of ethyl bromomethacrylate (9 mmol) in 20 mlof diethyl ether is added drop by drop. The solution is stirred for 30minutes at 0° C. and for 1 to 2 hours at ambient temperature. Thereaction mixture is diluted with 20 ml of water and the organic layer iswashed with water, and then dried on Na₂SO₄ and evaporated. Thecompounds 2h and 2i obtained in this way are purified by columnchromatography, using petroleum ether (40-60° C.)/Et₂O (8:2) as aneluent.

The yields are given in table 2.

EXAMPLE 10

In this example, the acrylate 2j wherein R³ corresponds to ROCH₂ andrepresents the group according to the formula C₆H₄—CH₂—O—CH₂ is preparedaccording to the reaction diagram illustrated above, with R OH.

In a completely dry flask, small pieces of sodium (10 mmol) are added to20 ml of dry diethyl ether. To this reaction mixture, a 10 mmol solutionof benzyl alcohol in 10 ml of diethyl ether is added over a 2 hourperiod, under a moderate reflux. The mixture is reflux boiled for 6additional hours. The white precipitate obtained is cooled in anice-bath, and then filtered, washed with dry and cold diethyl ether anddried on P₂O₅ to obtain the sodium salt with an 85 to 95% yield.

The reaction of said salt with ethyl bromomethacrylate and thepurification of the compound obtained are performed as described inexample 8.

The acrylate 2j obtained is listed in table 2.

EXAMPLES 11 TO 21

In these examples, phosphinic blocks 3 are prepared with R² and R³ givenin table 3, using the procedure described by Yiotakis et al, J. Org.Chem. 61, 1996, 6601-6605 [8].

This procedure firstly comprises the Michael reaction described in FIG.1. The initial phosphinic acid 1 (FIG. 1) was prepared according to themethod described by Baylis et al, J. Chem. Soc. Perkin Trans I, 1984, p.2845-2853 [13].

The acrylates 2 were prepared in examples 1 to 10. To prepare thephosphinic blocks, proceed as follows.

A suspension of 1 mmol of N-benzoyloxy-carbonylphosphinic acid 1 and 5mmol of hexamethyl-disilazane is heated to 110° C., for 1 hour, in anitrogen atmosphere.

1.3 mmol of the suitable acrylate 2 is then added drop by drop over a 15minute period. The reaction mixture is placed under stirring for 3additional hours at 110° C. The mixture is allowed to cool to 70° C. and3 ml of ethanol is added drop by drop. After cooling to ambienttemperature, the reaction mixture is concentrated. The residue ispurified by column chromatography using a chloroform/methanol/aceticacid (7:0.5:0.5) mixture as an eluent. In this way, the phosphinicblocks 3a to 3k are obtained with the yields given in table 3.

In this table, the R³ column indicates which acrylate 2 was used for thesynthesis of the phosphinic block. Table 3 also gives the Rf of theblocks obtained.

EXAMPLES 22 AND 23

In these examples, the phosphinic blocks 3l and 3m complying with theformulas given in table 3 are synthesised using the following procedure.

2.1 mmol of diisopropylamine and 2.1 mmol of trimethylsilyl chloride areadded to a solution chilled in ice of 1 mmol of phosphinic acid 1 in 2ml of chloroform.

The mixture is placed under stirring at ambient temperature for 3 hours.After cooling to 0° C., 1.4 mmol of suitable ethyl acrylate 2 (2e or 2g)is added, and the reaction mixture is stirred at ambient temperature for16 hours. After adding ethanol drop by drop, the solvents are eliminatedin a vacuum. The compounds 3l or 3m in table 3 are obtained after columnchromatography as for examples 11 to 21.

The yields and Rf values of the blocks obtained are given in table 3.

In addition, the blocks 3b, 3d and 3e to 3k were characterised withproton NMR, carbon 13 and phosphorus. The results obtained are appended.

EXAMPLE 24

In this example, the compounds 4a to 4m in FIG. 1 are prepared fromcompounds 3a to 3m according to the following procedure.

1 mmol of compound 3a to 3m and 1.2 mmol of 1-adamantyl bromide aredissolved in 10 ml of chloroform. The reaction mixture is reflux boiled.2 mmol of silver oxide is then added in five equal parts, over a 50minute period. The solution is reflux boiled for 30 more minutes. Afterthe solvents are eliminated, the residue is treated with diethyl etherand filtered on Celite. The filtrates are concentrated. The residue ispurified by column chromatography, using the chloroform/isopropanol(9.8:0.2) mixture as an eluent. In this way, the compounds 4a to 4m areobtained with the yields given in table 4, the Rf values of saidcompounds are also given in table 4.

EXAMPLE 25

In this example, the procedure described in FIG. 1 is followed toconvert compounds 4a to 4m into compounds 5a to 5m. For this purpose,the following procedure is used.

1 ml of 4N NaOH is added drop by drop to a stirred solution of 1 mmol ofcompound 4a to 4m in 5.5 ml of methanol. The reaction mixture is stirredfor 18 hours, and the solvent is then eliminated. The residue is dilutedwith water, and then acidified with 0.5 N HCl in an ice bath, dried onNa₂SO₄ and concentrated to obtain compounds 5a to 5m in white solidform, with the yields given in table 5. The Rf values of said compoundsare also given in table 5.

EXAMPLE 26

In this example, phosphinic pseudo-peptides wherein the formulas aregiven in tables 5 to 7 are prepared from the phosphinic blocks in table4, by means of Fmoc (fluorenylmethoxycarbonyl) chemistry solid phasesynthesis, according to the procedure described by Yotakis et al, 1996,J. Org. Chem. 61, pages 6601-6605 [8] and Jiracek et al in J. Biol.Chem., 1995, 270, pages 21701-21706 [9] and in J. Biol. Chem., 1996,271, pages 19606-19611 [10].

The coupling is carried out by means of the in situ strategy using2-(1H-benzotriazol-1-yl-1,1,3,3-tetramethyluronium-hexafluorophosphate(HBTU)/diiso-propylethylamine.The coupling conditions are as follows: three equivalents of amino acidFmoc derivative and 4 equivalents of diisopropylethylamine indimethylformamide are added to the resin and left to react for 30 min.For the coupling of the phosphinic blocks:

1.5 block equivalents are used.

The splitting conditions of the Fmoc group are 50% piperidine indimethylformamide for 30 minutes. The Fmoc group is (fluoremylmethoxy)carbonyl.

EXAMPLE 27

In this example, the pseudo-peptides given in table 8 comprisingdifferent types of R¹ group are synthesised. The R¹ group introductionconditions are given below, according to the type of group.

If R¹ is an acid comprising an indole or quinoline group (compounds 30,31, 32, 33 in table 8), the R¹ group was coupled on the peptide of thegeneric formula

still coupled to the resin, under the following conditions: 3equivalents of acid diluted in a small volume of N-methyl-pyrolidone, 3equivalents of HBTU (0.4 M), 10 equivalents of diisopropylethylamine(1.2 M), with a coupling time of 45 min. The incorporation of the R¹group is followed by a Kaiser test. When required, this operation wasrepeated several times, until the amine function was completelysubstituted.

The R¹═Ph—CH₂—O—CH₂—CO— (compound 26) group was incorporated using thecorresponding chlorinated derivative under the following conditions: thechlorinated derivative (25 equivalents, 0.5 M/dichloromethane) anddiisopropylethylamine (25 equivalents, 0.5 M/dichloromethane) are addedto the resin. The acylation reaction requires 1/2 h.

For the synthesis of compounds 27, 28 and 29, the peptide

still coupled to the resin, was firstly acylated with the Br—CH₂—CO—Brderivative under the following conditions: the brominated derivative (25equivalents, 0.5 M/dichloromethane) and diisopropylethylamine (25equivalents, 0.5 M/dichloromethane) are added to the resin, for areaction time of 1/2 h. The second step corresponds to the alkylation ofthe corresponding aniline derivatives. For this purpose, the anilinederivative (50 equivalents/DMSO) is added to the resin, for a couplingtime of 2.5 h.

The splitting of the peptides of the resin, and the hydrolysis of theprotective groups were carried out using a trifluoroacetic acid solutioncontaining 2.5% water, 2.5% thioanisoi, 1.25% thiophenol, 1.25%ethanedithiol and 1.25% triisopropylsilane.

All the peptides synthesised in examples 26 and 27 were purified byreverse phase HPLC using gradients produced with water, acetonitrilesolutions containing 0.01% trifluoroacetic acid. In the majority ofcases, four peaks are observed in the chromatograms, corresponding tothe four forms of diastereoisomers generated by the synthesis protocolof said phosphinic compounds. All the phosphinic peptides purified inthis way were inspected using mass spectroscopy.

EXAMPLE 28

In this example, the affinity constant Ki of the pseudo-peptides intables 5 to 8 are examined with respect to the different matrixmetalloproteases MMP.

The MMPs used were produced in recombinant form, in an E. coliexpression system, and then purified using different types ofchromatographies. Apart from stromelysin-3 (MMP-11), the MMPs producescorrespond to human sequences. The Stromelysin-3 used in this studycorresponds to the murine form.

The activity of each MMP was determined using two fluorogenic syntheticsubstrates. The cutoff of said substrates, which generates a fluorescentsignal proportional to the quantity of split substrate, enables aprecise determination of the kinetic parameters. The values of theconstants Km of the substrates, taken into account to determine the Kivalues, are given in the table below. The affinity constants Ki werecalculated using the equation of Horowitz et al, Proc. Natl. Acad. Sci.USA, 1987, 84, p. 6654-6658 [14], accounting for the dependency of thepercentage of inhibition measured experimentally according to theconcentration of inhibitor, as a function of the enzyme and substrateconcentration.

The experiments for conducted in a Tris/50 mM HCl buffer, pH 6.8, 10 mMCaCl₂, 25° C.

type of MPP Km μM MMP-11 0.2 MMP-2 58 MMP-9 13 MPP-14 26 MMP-1 93 MMP-786 MMP-8 60

The results obtained are given in tables 5 to 8.

The generic formula of the inhibitors synthesised in this case indicatesthat they are phosphinic pseudo-tripeptides. According to the assumedbonding mode of said compounds with the MMPs' active site, saidinhibitors must interact with the sub-sites S1, S1′ and S2′ of saidenzymes, respectively. The R1group comprised on most of the inhibitorsis assumed to interact with the junction of sub-sites S3/S2.

In table 5, the influence of the type of R³ residue on the efficacy ofthe pseudo-peptide as an MMP inhibitor.

The interaction of the inhibitors with the sub-site S1′, involving theR³ group of the compounds, was the subject of an in-depth study, sincethis sub-site is thought to mainly control enzyme-inhibitor interactionselectivity.

The analysis of table 5 demonstrates that the size and nature of the R³residue plays a critical role in the affinity of compounds: in this way,the activity of the inhibitors appears to be multiplied by a factor of30 in the case of MMP-8, when the R ³ group changes from a benzyl tophenylpropyl residue (compounds 7 and 9). It is noted that the gain inaffinity corresponding to this substitution is not constant for thedifferent MMPs, suggesting that the sub-site S1′ of each MMP may besensitive to the nature of the R³ residue. In this respect, it isinteresting to observe that MMP-11 is the only MMP to prefer phenethylresidue, in relation to a phenylpropyl residue (compound 8 and 9).

The comparison of compounds 9, 10 and 11 which only differ by a singleatom (C, O, S) on the R³ residue, suggests the existence of a specificinteraction between the sulphur atom in the γ position of R³ of theinhibitors and the sub-site S1′ of the MMPs. Irrespective of the type ofMMP, compound 11 is always the most powerful of these three inhibitors.

In table 6, the inhibitors are characterised by the presence of a phenylresidue in R², instead of a methyl residue (table 5). The comparison oftables 5 and 6 reveals that the presence of a benzyl residue makes itpossible to increase the overall affinity of the inhibitors. In thespecific case of MMP-11, the methyl->benzyl substitution induces a muchmore significant increase for this MMP, than for the others. This resultindicates the preference of the sub-site S1 of MMP-11 for a benzylresidue, in relation to a methyl residue.

Introducing a greater diversity in R³ in this series made it possible toobtain a clearer idea of the influence of the residue in R³. This seriesdemonstrates that, to obtain powerful phosphinic inhibitors with respectto certain MMPs, in the R³ position, it is necessary to introduce a longarylalkyl chain. In this way, compound 18 is an example of a verypowerful inhibitor of MMP-8, MMP-11, MMP-2 and MMP-9.

In relation to these MMPs, it is noted that the inhibitors reported inthis study appears to be less powerful with respect to MMP-14.

In addition, as a general rule, these pseudo-peptides appear to have alow activity with respect to MMP-1 and MMP-7.

It should be noted that compound 14 comprising a phenethyl R³ residueappears to be very powerful on MMP-11, but it is much less active onother MMPs.

Table 7 illustrates the importance of the tryptophan residue on the R⁴position, and the importance of the tryptophan residue configuration.Compound 24 indicates that the tryptophan in these inhibitors may bereplaced by another aromatic residue Dpa (dinitrobenzyl) in the case ofMMP-11 and MMP-8

Table 8 illustrates the effect of different modifications on the R¹group (Table IV). The analysis of the results indicates that the natureof R¹ influences the affinity of compounds with respect to differentMMPs. Compound 31 is an example of a very powerful inhibitor withrespect to MMP-11, showing a certain selectivity for said enzyme.Compounds 34 and 35 represent examples of powerful inhibitors wherein R¹corresponds to a natural amino acid.

EXAMPLE 29

In this example, the influence of the configuration of the two positionsR² and R³ on the efficacy of pseudo-peptides as an MMP inhibitor isstudied.

The synthesis process used in the above examples to prepare thephosphinic derivatives according to this patent produces each inhibitorin the form of a mixture of four diastereoisomers, due to the presenceto two asymmetric centres on the alpha carbon comprising the R² and R³residues. To evaluate the influence of the configuration of these twopositions, compound 15 was resynthesised using optically pure phosphinicphenylalanine amino acid, of an R or S configuration. Each synthesisproduces a mixture of two diastereoisomers:

Z-(S)PheY(PO₂CH₂) (S)pPhe-Trp-NH₂ and Z-(S)PheY(PO₂CH₂) (R)pPhe-Trp-NH₂or

Z-(R)PheY(PO₂CH₂) (S)pPhe-Trp-NH₂ and Z-(R)PheY(PO₂CH₂) (R)pPhe-Trp-NH₂

that can easily be separated by reverse phase HPLC. Table 9 shows theactivity of the four diastereoisomers of compound 15 with respect to thedifferent MMPs. It is interesting to note that for this class ofphosphinic compounds, at least three diastereoisomers inhibit thedifferent MMPs in an almost equivalent manner. This property, apart frombeing a means to control inhibitor selectivity, could also prove to bevery important in terms of metabolism and pharmacokinetics, twoparameters which could be sensitive to the molecular stereochemistry.

The results of the characterisation by proton NMR, carbon 13 andphosphorus of the RI fraction of compound 15 are appended.

EXAMPLE 30

In this example, the anti-tumour efficacy of compound 15 (RI fraction)in table 9 (RXPO3) is measured.

The tumorigenesis model used to test the effect of said RXPO3 inhibitorin vivo consists of a subcutaneous graft of malignant murine C26 cellsin syngenic mice (with a genetic background similar to that of theinjected cells).

The C26 cells established from Balb c mouse colic cancer (Corbett etal., 1975, Cancer Res, 35:2434-2439 [15] are cultured untilsubconfluence. After trypsinisation, the cells are centrifuged at 1000 gfor 10 minutes. The sediment is washed and resuspended in 1×PBS. A 200ml volume, containing 5×10⁴ C26 cells, is injected subcutaneously at 2sites on the back of 8 to 9-week old mice. The inhibitor is solubilisedin 1× PBS and 150 mg of inhibitor in a 150 ml volume is administered bythe intraperitoneal route. The treatment starts on the day of theinjection of the C26 cells and continues at a rate of one injection aday for 25 days. The tumoral volumes are measured every day.

Three identical and independent tumorigenesis experiments wereconducted, according to the protocol given above. The results obtainedwere similar. One of them is reported below.

The experiment related to 12 animals, 6 receiving a treatment (150 μg ofinhibitor/mouse/day) and 6 used as controlled animals (150 μl 1×PBS).FIG. 2 shows the median values of the tumoral volumes as a function ofthe time elapsed since the injection of the C26 cells (Days 10 to 25).

It is observed that the tumours start to appear on day D10, and thattheir mean and median volumes are always lower in the animals treatedwith the inhibitor RXPO3. This difference in tumour size is of the orderof 50% on day D15 to day D20. Subsequently, the efficacy of theinhibitor appears to be lower. These results are in line with the recentobservations made on another tumorigenesis model using wild or deficientanimals for the expression of stromelysin-3 (MMP-11) and demonstratingthat this matrix metalloprotease is involved in the initial implantationsteps and favours the development of tumours. According to this model,the efficacy of the inhibitors must be maximal during the first days oftumour development.

References

[1]: Brown, Medical Oncology, 1997, 14, p. 1-10.

[2]: Beckett et al, 1998, Exp. Opin. Ther. Patents, 8, p. 259-289.

[3]: Goulet et al, Bioorg. Med. Chem. Lett. 4, 1994, p. 1221-1224.

[4]: Caldwell et al, Bioorg. Med. Chem. Letter 6, 1996, p.323-328.

[5]: FR-A-2 676 059.

[6]: FR-A-2 689 764.

[7]: EP-A-0 725 075

[8]: Yotakis et al, J. Org. Chem., 1996, 61, p. 6601-6605.

[9]: Jiracek et al, J. Biol. Chem., 1995, 270, p. 21701-21706.

[10]: J. Biol. Chem., 1996, 271, p. 19606-19611.

[11]: Eistetter et al, J. Med. Chem., 25, p 109-113, 1982.

[12]: Baldwin. et al, J. Chem. Soc. Chem. Comm. 1986, p 1339-1340.

[13]: Baylis et al, J. Chem. Soc. Perkin Trans I, 1984, p. 2845-2853.

[14]: Horowitz et al, Proc. Natl. Acad. Sci. USA, 1987, 84, p.6654-6658.

[15]: Corbett et al., 1975, Cancer Res, 35:2434-2439.

TABLE 1 Acrylate according to the formula:

com- Pf (mm Hg) Yield Ex. pound R³ ° C. (%) 1 2a C₆H₅CH₂— 64-65 80 (0.01) 2 2b C₆H₅—(CH₂)₂— 90-95 76 (0.7) 3 2c C₆H₅(CH₂)₃— 112-115 87(0.6) 4 2d C₆H₅(CH₂)₄— 120-125 78  (0.005) 5 2e

Oil 25 6 2f CH₃(CH₂)₆— 101-105 40 (8) 7 2g

Oil 13

TABLE 2 Acrylate according to formula:

Ex. compound R³ = R X CH₂ where X = O or S. Yield 8 2h

80% 9 2i

88% 10 2j

85%

TABLE 3 Phosphinic Yield Ex. block R² R³ % Rf 11 3a H C₆H₅CH₂ 80 0.41(2a) 12 3b H C₆H₅—(CH₂)₂ 95 0.47 (2b) 13 3c H C₆H₅(CH₂)₃ 83 0.57 (2c) 143d H C₆H₅—CH₂—O—CH₂ 61 0.69 (2j) 15 3e H C₆H₅—CH₂—S—CH₂ 60 0.48 (2i) 163f Phenyl C₆H₅(CH₂)₂ 85 0.77 (2b) 17 3g ″ C₆H₅(CH₂)₃ 79 0.80 (2c) 18 3h″ C₆H₅(CH₂)₄ 75 0.82 (2d) 19 3i ″ CH₃(CH₂)₆ 81 0.68 (2f) 20 3j ″C₆H₅—CH₂—O—CH₂ 64 0.61 (2j) 21 3k ″

40 0.63 22 31 ″

60 0.66 23 3m ″

30 0.65

TABLE 4 Initial Compound Compound compound 4 Yield Rf¹ 5 Yield Rf² 3a 4a95 0.70 5a 80 0.53¹ 3b 4b 97 0.72 5b 81 0.59¹ 3c 4c 97 0.73 5c 85 0.213d 4d 93 0.77 5d 90 0.42¹ 3e 4e 72 0.72 5e 92 0.50¹ 3f 4f 89 0.61 5f 950.42 3g 4g 75 0.66 5g 97 0.42 3h 4h 95 0.63 5h 95 0.44 3i 4i 96 0.78 5i78 0.56 3j 4j 91 0.71 5j 80 0.26 3k 4k 93 0.66 5k 94 0.46 3l 4l 91 0.535l 92 0.38 3m 4m 95 0.57 5m 86 0.35 ¹in the hexane/ethyl acetate/aceticacid (3:3:0.2) mixture ²in the chloroform/methanol (9.5:0.5) mixture.

TABLE 5 Influence of the R³ group: Value of affinity constants Ki.(XVIII)

Com- pound MMP-11 MMP-2 MMP-9 MMP-14 MMP-1 MMP-7 MMP-8 No [R3] = mST3Gel-A Gel-B MT1-MMP HFC Matrilysin HNC 7 CH₂-φ (Phe) 350 nM 250 nM 280nM  2030 nM  24% @ 2 mM 71% @ 0 mM  240 nM  8 CH₂—CH₂-f  51 nM  80 nM 60nM 270 nM 23% @ 2 mM 43% @ 50 mM 20 nM 9 CH₂—CH₂—CH₂-f 100 nM  31 nM 23nM  92 nM 30% @ 2 mM 51% @ 50 mM  8 nM 10 CH₂—O—CH₂-f 175 nM 250 nM 44nM 550 nM 15% @ 2 mM 27% @ 50 mM 19 nM 11 CH₂—S—CH₂-f  36 nM  14 nM  6nM  26 nM 45% @ 2 mM 36% @ 50 mM <0.5 nM

TABLE 6 Influence of the R³ group: Value of affinity constants Ki. (XIX)

[R3] = MMP-11 MMP-2 MMP-9 MMP-14 MMP-1 MMP-7 MMP-8 12 CH₃ (Ala) 2670 nM 0% @ 2 mM 0% @ 2 mM 0% @ 10 mM 20% @ 2 mM 10% @ 50 mM 9% @ 1 mM 13CH₃—CH—(CH₃)₂ (Leu) 22 nM 202 nM 65 nM 192 nM 45% @ 2 mM 210 nM 40 nM 14CH₂—CH₂-φ 8.8 nM  275 nM 110 nM  660 nM 10% @ 2 mM 62% @ 50 mM 45 nM 15CH₂—CH₂—CH₂-φ  5 nM  20 nM 10 nM 105 nM 23% @ 2 mM 65% @ 50 mM 2.5 nM 16 CH₂—CH₂—CH₂—CH₂-φ 33 nM 145 nM 70 nM 580 nM  5% @ 2 mM 65% @ 50 mM4.3 nM  17 CH₂—O—CH₂-φ 16 nM  85 nM 55 nM 545 nM  9% @ 2 mM 40% @ 50 mM20 nM 18 CH₂—S—CH₂-f-OMe  2 nM  6 nM  3 nM  22 nM 13% @ 2 mM 84% @ 50 mM0.7 nM  19 CH₂-Naphthyl 74 nM 330 nM 675 nM  1350 nM   0% @ 5 mM 1800 nM230 nM  20 CH₂—CH₂-Naphthyl 12 nM  30 nM 55 nM 125 nM  0% @ 2 mM  210 nM34 nM 21 (CH₂)₇ 34 nM  75 nM 30 nM 271 nM 27% @ 2 mM 50% @ 50 mM  6 nM

TABLE 7 Influence of residue in Yaa′ position: Value of affinityconstants Ki. (XX)

Compound MMP-11 MMP-2 MMP-9 MMP-14 MMP-1 MMP-7 MMP-8 No. Yaa′ mST3 Gel-AGel-B MT1-MMP HFC Matrilysin HNC 22 Ala 20% @ 1 mM 31% @ 2 mM 45% @ 2 mM2960 nM 9% @ 2 mM 23% @ 50 mM 240 nM 15 L-Trp 5 nM 20 nM 10 nM 105 nM23% @ 2 mM 65% @ 50 mM 2.5 nM 23 D-Trp 1% @ 1 mM 6% @ 2 mM 14% @ 2 mM10% @ 10 mM 3% @ 10 mM 18% @ 50 mM 2% @ 1 mM 24 Dpa 27 nM 260 nM 245 nM1282 nM 9% @ 2 mM 61% @ 50 mM 25 nM

TABLE 8 Influence of R1 residue: Value of affinity constants Ki. (XXI)

Compound MMP-11 MMP-2 MMP-9 MMP-14 MMP-1 MMP-7 MMP-8 No. [R1] = mST3Gel-A Gel-B MT1-MMP HFC Matrilysin HNC 25

20 nM 26 nM 35 nM 90 nM 29% @ 1 μM 16% @ 1 μM 3.5 nM 26

5 nM 8 nM 10 nM 40 nM 28% @ 1 μM 33% @ 1 μM 2.5 nM 27

15 nM 17 nM 6 nM 73 nM 33% @ 1 μM 16% @ 1 μM 4.5 nM 28

3.8 nM 9 nM 6 nM 45 nM 63% @ 1 μM 25% @ 1 μM 4 nM 29

10 nM 30 nM 34 nM 63 nM 55% @ 1 μM 48% @ 1 μM 7.5 nM 30

1.5 nM 10 nM 8 nM 41 nM 87% @ 1 μM 605 nM 1? nM 31

0.9 nM 24 nM 7 nM 32 nM 36 nM 117 nM 5 nM 32

4.2 nM 19 nM 13 nM 60 nM 78% @ 1 μM 370 nM 5 nM 33

5 nM 100 nM 110 nM 217 nM 47% @ 1 μM 37% @ 1 μM 17 nM 34 Z-Ala 8 nM 11nM 10 nM 41 nM 24% @ 1 μM 18% @ 1 μM 5.5 nM 35 Z-Leu 6 nM 40 nM 22 nM 53nM 20% @ 1 μM 32% @ 1 μM 7 nM

TABLE 9 influence of R² and R³ residue configuration: Value of affinityconstants Ki. (XXII)

R2 residue HPLC MMP-11 MMP-2 MMP-9 MMP-14 MMP-1 MMP-7 MMP-8configuration fraction mST3 Gel-A Gel-B MT1-MMP HFC Matrilysin HNC S I 5nM 45 nM 33 nM 135 nM 17% @ 2 mM 59% @ 20 mM 16 nM S II 50 nM 365 nM 250nM 2290 nM 6% @ 2 mM 29% @ 20 mM 460 nM R I 6 nM 54 nM 42 nM 90 nM 18% @2 mM 44% @ 20 mM 16 nM R II 9 nM 42 nM 78 nM 385 nM 16% @ 2 mM 34% @ 20mM 65 nM

Block 3b: ZAlaΨ(PO₂CpH₂)hPheOEt. ¹H Z Phe hPhe OEt NH 5.36 Hα 4.05 2.85Hβ 1.28 1.95/1.84 Hγ 2.55 CH₃ 1.28 Arom 6.62-7.33 6.62-7.33 CH₂/CpH₂5.12 1.84/2.25 4.1 ¹³C Z Phe hPhe OEt Cα 45.35/46.05* 39.2 Cβ 14.6 35.9Cγ 33.4 CH₃ 61.4 Arom   126.4-141.4 126.4-141.4 C = 0 156.2 174.9 CH₂/Cp67.6  28.5/27.8* 14.6 *2 diastereoisomers

δ(ppm) Ref. ¹H CHCl₃ (7.26 ppm). Temp.: 298 K. Solvent: CDCl₃ Ref. ¹³CCDCl₃ (77.36 ppm). Ref. ³¹P H₃PO₄ (0 ppm). Block 3d:ZAlaΨ(PO₂CpH₂)Ser(Bn)OEt. ¹H Z Ala Ser(Bn) OEt NH 5.41 Hα 4.05 3.09 Hβ1.35 3.65 Hδ 4.48 CH₃ 1.2 Arom 7.26-7.32 7.26-7.32 CH₂/CpH₂ 5.092.02/2.25 4.12 ¹³C Z Ala Ser(Bn) OEt Cα 45.20/46.8* 40.13 Cβ 14.42 71.38Cδ 73.5 CH₃ 61.77 Arom 127.9-138.2 127.9-138.2 C = 0 156.3 173.2 CH₂/Cp67.64  24.16/25.77* 14.42 *2 diastereoisomers

δ(ppm) Ref. ¹H CHCl₃ (7.26 ppm). Temp.: 298 K. Solvent: CDCl₃ Ref. ¹³CCDCl₃ (77.36 ppm). Ref. ³¹P H₃PO₄ (0 ppm). Block 3e:ZAlaΨ(PO₂CpH₂)Cys(Bn)OEt. ¹H Z Ala Cys(Bn) OEt NH 5.41 Hα 4.08 3 Hβ 1.352.50/2.75 Hδ 3.65 CH₃ 1.22 Arom 7.26-7.33 7.26-7.33 CH₂/CpH₂ 5.122.02/2.25 4.15 ¹³C Z Ala Cys(Bn) OEt Cα 44.93/46.7* 39.19 Cβ 14.4 34.56Cδ 36.33 CH₃ 61.7 Arom 127.4-138.1 127.4-138.1 C = 0 156.17 173.56CH₂/Cp 67.78  26.88/28.19* 14.43 *2 diastereoisomers

δ(ppm) Ref. ¹H CHCl₃ (7.26 ppm). Temp.: 298 K. Solvent: CDCl₃ Ref. ¹³CCDCl₃ (77.36 ppm). Ref. ³¹P H₃PO₄ (0 ppm). Block 3f:ZPheΨ(PO₂CpH₂)hPheOEt. ¹H Z Phe hPhe OEt NH 5.61 Hα 4.2 2.92 Hβ3.17/2.83 1.97/1.83 Hγ 2.55 CH₃ 1.23 Arom 7.0-7.3 7.0-7.3 CH₂/CpH₂ 51.85/2.22 4.15 ¹³C Z Phe hPhe OEt Cα  52.1/53.5* 39.8 Cβ 34 35.5 Cγ 33.1CH₃ 63.05 Arom 127-141 127-141 C = 0 158 177.6 CH₂/CpH₂ 68.5  28.9/27.7*14.1 *2 diastereoisomers

δ(ppm) Ref. ¹H CHCl₃ (7.26 ppm). Temp.: 298 K. Solvent: CDCl₃ Ref. ¹³CCDCl₃ (77.36 ppm). Ref. ³¹P H₃PO₄ (0 ppm). Block 3g:ZPheΨ(PO₂CpH₂)pPheOEt. ¹H Z Phe pPhe OEt NH 4.98 Hα 4.2 2.87 Hβ3.23/2.83 1.53/1.45 Hγ 1.51 Hδ 2.52 CH₃ 1.21 Arom 7.14-7.26 7.14-7.26CH₂/CpH₂ 4.94 1.75-2.21 4.13 ¹³C Z Phe pPhe OEt Cα 51.75/53.3* 39.7 Cβ33.85 33.6 Cγ 28.4 Cδ 35.7 CH₃ 62.3 Arom 126.6-142.3 126.6-142.3 C = 0157.7 177.2 CH₂/Cp 68.3  29.1/28* 13.9 *2 diastereoisomers

δ(ppm) Ref. ¹H CHCl₃ (7.26 ppm). Temp.: 298 K. Solvent: CDCl₃ Ref. ¹³CCDCl₃ (77.36 ppm). Ref. ³¹P H₃PO₄ (0 ppm). Block 3h:ZPheΨ(PO₂CpH₂)bPheOEt. ¹H Z Phe bPhe OEt NH 5.4 Hα 4.25 2.8 Hβ 3.26/2.821.62/1.53 Hγ 1.25 Hε 1.52 Hδ 2.52 CH₃ 1.2 Arom  7.0-7.26  7.0-7.26CH₂/CpH₂ 4.99 1.75-2.23 4.1 ¹³C Z Phe bPhe OEt Cα  52.2/50.9* 39.7 Cβ34.4 34 Cγ 26.4 Cδ 31.2 Cε 35.8 CH₃ 61.4 Arom 126.4-142.6 126.4-142.6 C= 0 156.5 175.3 CH₂/Cp 67.45 28.44/29.3* 14.2 *2 diastereoisomers

δ(ppm) Ref. ¹H CHCl₃ (7.26 ppm). Temp.: 298 K. Solvent: CDCl₃ Ref. ¹³CCDCl₃ (77.36 ppm). Ref. ³¹P H₃PO₄ (0 ppm). Block 3i:ZPheΨ(PO₂CpH₂)HeptOEt. ¹H Z Phe Hept OEt NH 5.46 Hα 4.26 2.8 Hβ3.28/2.85 1.50/1.63 Hγ-Hζ 1.2 CH₃ 0.83 1.23 Arom 7.16/7.27 7.67-7.27CH₂/CpH₂ 4.98 1.75/2.23 4.2 ¹³C Z Phe Hept OEt Cα 51.25/52.8* 39.31 Cβ34.2 34.6 Cγ/Cζ 31.97 · 29.31 26.65 · 22.81 CH₃ 14.5 61.2 Arom127.5-137.6 127.5-137.6 C = 0 156.36 175.37 CH₂/Cp 67.23  29.73/28.45*14.6 *2 diastereoisomers

δ(ppm) Ref. ¹H CHCl₃ (7.26 ppm). Temp.: 298 K. Solvent: CDCl₃ Ref. ¹³CCDCl₃ (77.36 ppm). Ref. ³¹P H₃PO₄ (0 ppm). Block 3j:ZPheΨ(PO₂CpH₂)Ser(Bn)OEt. ¹H Z Phe Ser(Bn) OEt NH 5.48 Hα 4.3 3.12 Hβ3.28/2.85 3.65 Hδ 4.45 CH₃ 1.22 Arom 7.16-7.36 7.16/7.36 CH₂/CpH₂ 4.97 2.0/2.27 4.15 ¹³C Z Phe Ser(Bn) OEt Cα 52.65/50.9* 39.93 Cβ 34.16 71.42Cδ 73.44 CH₃ 61.6 Arom   126-138.1   126-138.1 C = 0 156.4 173.2 CH₂/Cp67.47  24.9/25.36* 13.94 *2 diastereoisomers

δ(ppm) Ref. ¹H CHCl₃ (7.26 ppm). Temp.: 298 K. Solvent: CDCl₃ Ref. ¹³CCDCl₃ (77.36 ppm). Ref. ³¹P H₃PO₄ (0 ppm). Block 3k:ZPheΨ(PO₂CpH₂)Cys(pOMeBn)OEt. ¹H Z Phe Cys(pOMeBn) OEt NH 5.41 Hα 4.33.03 Hβ 3.27/2.80 2.53/2.70 Hγ 3.6 CH₃ 3.75 1.2 Arom 7.18-7.36 7.18-7.36CH₂/CpH₂ 4.98 2.03/2.15 4.15 ¹³C Z Phe Cys(pOMeBn) OEt Cα  52.1/50.8*39.6 Cβ 34.42 35 Cδ 35.6 CH₃ 55.98 61.74 Arom 127.2-130.4 127.2-130.4 C= 0 159 173.6 CH₂/Cp 67.32  27.9/29.1* 14.46 *2 diastereoisomers

δ(ppm) Ref. ¹H CHCl₃ (7.26 ppm). Temp.: 298 K. Solvent: CDCl₃ Ref. ¹³CCDCl₃ (77.36 ppm). Ref. ³¹P H₃PO₄ (0 ppm). Compound 15:ZPheΨ(PO₂CpH₂)pPheTrpNH₂ RI fraction in table 9 ¹H Z Phe pPhe Trp NH5.63 7.05 Hα 4.17 2.87 4.72 Hβ 3.13/2.75 1.59/1.40 3.25 Hγ 1.45 Hδ 2.46CH₂/CpH₂ 4.95 1.81/2.06 ¹³C Z Phe pPhe Trp Cα 52 54.7 Cβ 33.85 34.0627.7 Cγ 28.53 Cδ 35.63 C = 0 158.17 177.19 176.93 CH₂/Cp 68.81 28.45

δ(ppm) Ref. ¹H CHCl₃ (7.26 ppm). Temp.: 298 K. Solvent: CDCl₃ Ref. ¹³CCDCl₃ (77.36 ppm). Ref. ³¹P H₃PO₄ (0 ppm).

What is claimed is:
 1. Phosphinic pseudo-peptide according to the formula:

wherein R¹ is an amine-protecting group, R² represents the lateral chain of a natural amino acid or an analogue thereof, R³ represents: 1) the lateral chain of a natural amino acid except for Gly and Ala, not substituted or substituted by an aryl group, 2) an aralkyl group, or 3) an alkyl group comprising at least 3 carbon atoms, and R⁴ represents the lateral chain of a natural amino acid or an analogue thereof.
 2. Pseudo-peptide according to claim 1, wherein R² represents a methyl or benzyl group.
 3. Phosphinic pseudo-peptide according to the formula:

wherein R¹ is an amine-protecting group, R² represents the lateral chain of a natural amino acid or an analogue thereof, R³ represents the lateral chain of an amino acid selected from the group consisting of Phe, Leu, Ser and Cys residue, wherein the lateral chain is substituted by an aralkyl group, and R⁴ represents the lateral chain of a natural amino acid or an analogue thereof.
 4. Pseudo-peptide according to claim 1, wherein R³ is an aralkyl group selected from the groups according to the formula:

where n is an integer from 1 to 4, and according to the formula:

where p is equal to 1 or
 2. 5. Pseudo-peptide according to claim 1 wherein R³ is a group complying with any of the following formulas:


6. Pseudo-peptide according to claim 1, wherein R³ is the group according to the formula:


7. Pseudo-peptide according to claim 1 wherein R⁴ represents the group according to the formula:


8. Pseudo-peptide according to claim 1, wherein R¹ represents a group selected from the group consisting of acetyl, benzyloxyacetyl, phenylaminoacetyl, (m-chloro-phenyl)aminoacetyl, (2-hydroxy-5-chloro-phenyl) amino acetyl, indolyl-2-carbonyl, 4,6-dichloro-indolyl-2-carbonyl, quinolyl-2-carbonyl and 1-oxa-2,4-dichloro-7-naphthalene carbonyl groups.
 9. Pseudo-peptide according to claim 1, which complies with the formula:

wherein Z is a benzyloxycarbonyl group.
 10. Pseudo-peptide according to claim 9, wherein the unit

has an L configuration.
 11. A method for inhibiting at least one matrix zinc metalloprotease in a subject in need thereof, comprising administering to said subject a pharmaceutical formulation comprising at least one pseudo-peptide according to claim
 1. 12. Method according to claim 11, wherein the matrix zinc metalloprotease is selected from the group consisting of MMP-2, MMP-8, MMP-9 and MMP-11.
 13. Method according to claim 11, wherein said inhibiting is for the treatment of a disease characterised by the over-expression of matrix proteases.
 14. Method according to claim 13, wherein the disease is cancer.
 15. Method according to claim 11, wherein the pseudo-peptide complies with the formula:

wherein Z is a benzyloxycarbonyl group.
 16. Method according to claim 15, wherein the unit:

has an L configuration.
 17. Pseudo-peptide according to claim 1, wherein R¹ represents a group selected from the group consisting of t-butoxycarbonyl, benzyloxycarbonyl, cinnamoyl, pivaloyl, and N-(1-fluorenyl-methoxycarbonyl) Fmoc groups.
 18. Method according to claim 12, wherein said inhibiting is for the treatment of a disease characterised by the over-expression of matrix proteases.
 19. Method according to claim 18, wherein the disease is cancer.
 20. A pharmaceutical composition comprising at least one pseudo-peptide according to claim 1, in a therapeutically effective amount, and a pharmaceutically acceptable carrier.
 21. Phosphinic pseudo-peptide according to claim 1, wherein R⁴ represents a dinitrobenzyl group.
 22. Phosphinic pseudo-peptide according to claim 2, wherein R⁴ represents a dinitrobenzyl group. 